Research Article AppleCanActasAnti-AgingonYeastCells · and stationary (aged) cells grown in the...

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2012, Article ID 491759, 8 pagesdoi:10.1155/2012/491759

Research Article

Apple Can Act as Anti-Aging on Yeast Cells

Vanessa Palermo,1 Fulvio Mattivi,2 Romano Silvestri,3 Giuseppe La Regina,3

Claudio Falcone,1 and Cristina Mazzoni1

1 Dipartimento di Biologia e Biotecnologie Charles Darwin, Istituto Pasteur-Fondazione Cenci Bolognetti,Sapienza Universita di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy

2 Food Quality and Nutrition Department, Research and Innovation Centre, Fondazione Edmund Mach, Via E. Mach 1,38010 San Michele all’Adige, Italy

3 Dipartimento di Chimica e Tecnologie del Farmaco, Istituto Pasteur-Fondazione Cenci Bolognetti,Sapienza Universita di Roma, Piazzale Aldo Moro 5, 00185 Roma, Italy

Correspondence should be addressed to Cristina Mazzoni, [email protected]

Received 31 May 2012; Revised 7 July 2012; Accepted 14 July 2012

Academic Editor: Paula Ludovico

Copyright © 2012 Vanessa Palermo et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

In recent years, epidemiological and biochemical studies have shown that eating apples is associated with reduction of occurrenceof cancer, degenerative, and cardiovascular diseases. This association is often attributed to the presence of antioxidants such asascorbic acid (vitamin C) and polyphenols. The substances that hinder the presence of free radicals are also able to protect cellsfrom aging. In our laboratory we used yeast, a unicellular eukaryotic organism, to determine in vivo efficacy of entire apples andtheir components, such as flesh, skin and polyphenolic fraction, to influence aging and oxidative stress. Our results indicate thatall the apple components increase lifespan, with the best result given by the whole fruit, indicating a cooperative role of all applecomponents.

1. Introduction

During the last years, consumption of fruits and vegetableshas received growing interest because many epidemiologicaland biochemical studies have demonstrated that they possessbeneficial effects on human health. It is assumed that thecultivation of the apple tree goes back to prehistoric timesand has developed with the formation of the M. × domesticagene pool from M. sieversii, originary of Thien Shan region,in Central Asia [1].

Apples can be eaten in countless ways, such as fresh andas juice that can be processed into cider, apple vinegar, ordistilled.

Apples also contain nutrients and other compounds ofinterest, including high levels of polyphenols [2]. Thesecompounds have biological activity and they can influencedifferent mechanisms of fundamental relevance in cancerprevention [3]. The peel also contains high levels of triter-penoids, whereas red apples contain anthocyanins [4, 5].

The most studied molecules of this fruit are phenolic com-pounds that are distributed in the skin, in the core, and in theflesh. The composition of these compounds is quite variabledepending on the environment of growth, period, and yearof harvest. Generally, the average total polyphenol contentof the most commonly consumed apples varies between0.66 g/kg and 2.1 g/kg of fresh weight [6]. Apple polyphenols,and in particular the oligomeric proanthocyanidins, givethe largest contribution to the antioxidant activity of appleextracts [7].

Numerous studies undertaken in recent years have shownthat apples and their derivatives may have a wide range ofbiological activities, particularly useful in cases of asthma,cardiovascular disorders, polmonar disfunction and cancer.Studies in human models have shown that apples extractsmay have a role in preventing several types of cancer. It hasalso been amply demonstrated beneficial effects on aging inmammalian skin [8].

2 Oxidative Medicine and Cellular Longevity

Apple (sp. Malus domestica), is also rich in minerals,vitamin B, citric, and malic acid that can contribute to thewelfare of the body, promoting digestion and maintainingthe acidity of the digestive system.

It has been proposed that anticancer action of apple isdue to the presence of vitamin C and pectin, which, duringits fermentation, produce butyric acid, a substance used insome experimental drugs to treat cancer [9].

It was also shown that polyphenolic-rich apple extractsmay inhibit the activity of cytosolic PKC and have a rolein the suppression of human cancer cell growth in vitro [8]indicating that apple’s polyphenols could be of interest incancer prevention [2, 3].

Yeast has been extensively used as a model organismfor the study of complex phenomena that occur in highereukaryotes. The use of yeast compared to mammalian cellculture, lies in the ease of manipulation and the large degreeof homology between yeast and human genes.

Yeast is also used as a model to study aging as theprincipal pathways are conserved during evolution [10]. Inrecent years, several homologues of classical cell-death regu-lators have also been identified and characterized and yeastmutants that show an accelerated/delayed aging associatedwith early/late cell death are available to study the effect ofmolecules [11–13] on cell proliferation.

To test the effect of a substance on aging, cells aremaintained in the presence of the substance to be tested andtheir ability to form colonies over the time is measured.

An increase in cell viability in cultures containing asubstance, compared to the control, is a clear indicator of theeffectiveness of such substance on the aging process.

To make a more sensitive analysis, in this work we used apeculiar mutant (Kllsm4Δ) that shows premature aging andcell death [14–16]. We cultivated such strain in the presenceof apple extracts from skin, flesh, the entire fruit and thepartially purified polyphenols fraction and we found thatall of these components were able to increase lifespan atdifferent extents, with the best results given by the wholefruit, indicating that the latter contains additional importantsubstances. In view of future practical applications, we alsoverified the effectiveness of apple solutions included inointment, pointing to this kind of preparation as a base forthe prevention of skin aging.

2. Results

2.1. The Antiaging Power of Apples. To better understand theantiaging effects of apples, we followed the chronologicalaging of a Saccharomyces cerevisiae mutant that shows a verypremature aging [16] in the presence of apple raw extractsfrom the entire fruit, flesh, skin and also in the presence of apurified polyphenols fraction. This mutant, named Kllsm4Δ,is mutated in the LSM4, a key gene of mRNA degradation,and shows a marked stabilization of mRNA accompanied byrapid loss of viability during aging [14, 17, 18].

The comparison of the viability of the cells treatedwith different apple’s extracts with the control cultures, canfurnish a clear measure of their effectiveness on the agingprocess.

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Figure 1: Apple components can recover premature aging of ayeast early-aging mutant. Cells of strain MCY4/Kllsm4::KanMX4were grown in synthetic dextrose medium (SD) in the presence ofextracts from whole apple, skin, flesh, and polyphenolic fractions.Acetone 7% was used as a control. Viability was monitoredas percentage of microcolony forming units (CFU). Standarddeviation was obtained from three independent experiments.

Apple’s extracts consisting of freshly prepared, cen-trifuged and sterile filtered apple juice resuspended into70% acetone (See Section 4) were added, prior ten timedilution, to Kllsm4Δ1 cultures at the concentration of about26 mg/mL, and the chronological aging during stationaryphase of yeast samples was followed for 21 days.

As shown in Figure 1, the death kinetics of the Kllsm4Δ1strain grown in synthetic medium containing only 7%acetone is much faster compared to the cultures containingapple extracts. This difference, clearly evident since the earlydays, increases with time and we observed the complete lossof viability of control cultures after ten days of treatment.

In contrast, cultures containing the extracts from fleshand peel completely lost viability after 11 and 14 days,respectively, while in the presence of the whole apple extractviability ceased after twenty-one days, with an extensionof more than 100% compared to control cultures. Alsopolyphenols extracts exerted a marked effect in prolonginglifespan, conferring an extension of viability of 60%.

It has been suggested that reactive oxygen species (ROS)are the main cause of cellular aging as they can damage andcross-link DNA, proteins, and lipids.

For this reason, we looked by dihydrorhodamine (DHR)123 staining at the accumulation of ROS in yeast exponentialand stationary (aged) cells grown in the presence of applecomponents.

As previously demonstrated [19] and shown in Figure 2,DHR staining revealed that in the absence of externaloxidative stress, Kllsm4Δ1 cells accumulated ROS duringexponential phase (a) which increased with aging (b). Thepresence of apple components prevented ROS accumulation

Oxidative Medicine and Cellular Longevity 3

in both exponential and stationary phase cells, with theexception of the apple flesh.

This component, unexpectedly, seems to increase theproduction of ROS since the percentage of cells showing ROSwas higher than in control cultures both in exponential andstationary phase (Figure 2(c)).

We then checked the effect of apple extracts on cellsurvival following an oxidative stress such as the treatmentof cultures with hydrogen peroxide. The presence of fleshor skin extracts could not protect cells from such a stressin that viability was comparable to that of control cultures(Figure 3), and, at the concentration of 0,8 M H2O2, viabilitywas unclearly even lower.

On the contrary, both the polyphenols and the wholeextracts conferred to the cells higher resistance to hydrogenperoxide treatment. As shown in Figure 3, at the highestH2O2 concentration tested (3 mM) cells of control culturesshowed a viability of 15%, while in the presence of wholeapple and polyphenolic extracts showed a viability of28% and 20%, respectively, suggesting that these extractscontained antioxidant activity having an important role inpreventing damage from oxidative stress.

Then we wanted to confirm the results obtained withthe purified extracts using formulation containing a solutionfrom homogenates of flesh, skin, or the whole apple addedto ointment base. This kind of ointment might represent thebasis for a cosmetic use of apple for skin care.

We first evaluated the chronological aging of 10 mLyeast cultures grown in the presence of 1 gr of formulationcontaining the apple solutions. As can be seen in Figure 4(a),compared to the control, flesh and peel creams extendedlifespan of about 7% and 28,5%, respectively. The most strik-ing result was obtained with the whole apple homogenatethat extended cell viability of about 78,5%.

We also performed an additional test (viability spotassay) based on serial dilutions of treated cultures followedby cell plating on rich medium containing glucose as carbonsource. We incubated the plates at 28◦C and we thenfollowed the growth of strain Kllsm4Δ1 for 15 days on platescontaining rich medium (YPD) at intervals of three days. Asshown in Figure 4(b) after 20 days of growth at 28◦C, only theculture containing the whole apple survived better until thethird dilution, confirming the data on chronological aging.

We also evaluated the capacity of the creams containingthe different apple components to contrast the oxidativestress induced by treatment with hydrogen peroxide.

As shown in Figure 4(c), the cream added with thewhole apple solution showed the best protective effect againstoxidative stress in the presence of 0.8 mM H2O2 while, atincreasing concentrations the compound, cell grown withwhole apple and skin containing creams showed the highestprotection.

The apple extracts, which were prepared containing asimilar concentration of apple (see Section 4), clearly differin their concentration of total polyphenols, ranging from aminimum (ca. 138 mg/l) in apple flesh and in the partiallypurified polyphenols, to intermediate values (189 mg/l) inthe whole apple extract, up to much higher concentration(584 mg/l) in the apple skin extract. In conclusion, the total

polyphenols ranged in our extracts as follows: skin � wholeapple > flesh.

The apple extracts were screened also with an untar-geted LCMS method, in order to provide an unbiased,semiquantitative fingerprint of their composition. Thesedata can be useful to estimate the relative concentrationof known compounds, as well as for extracting additionalinformation useful for the design of future metabolomicsexperiment. The major apple phenolics according to theliterature [6] were assessed and their presence in the extractsis reported in Figure 5. The extracts were screened for thepresence of the most representative flavanols (catechin andepicatechin), procyanidin (procyanidin B2), dihydrochal-cones (phloridzin), cynnamic acids (chlorogenic acid), andflavonols (quercetin glycosides).

The qualitative composition of these extracts wasexpected to vary in light of the specific localization ofapple polyphenols in the different tissues [20]. In GoldenDelicious apple the quercetin glycosides, and in particularthe quercetin hexosides, are more concentrated in theskin cell layers lying just below the cuticle, while in thepericarp they are located mainly in correspondence with thevascular system, and the core is relatively rich in quercetin-rhamnoside [21]. Once the not edible part (the core) isremoved, the apple flesh is almost devoid of flavonols(Figure 5). Also the procyanidins B2, are more concentratedin the cell layers, ca. 150 micron thick, just below the cuticlewhile its concentration decreases inside the fruit [21] andthis is reflected in the composition of the extract in flavonols(Figure 5).

In summary and in agreement with the literature, appleflavonols were almost absent in the flesh extract, and ca. 3times more concentrated in skin extract in respect of bothwhole apple and purified polyphenols extracts. The majorflavonols (epicatechin and procyanidin B2), and phloridzinwere 2 times higher in skin versus the other extracts. Bothcatechin and chlorogenic acid were contained in similarconcentrations in all the extracts, including the flesh.

3. Discussion

Besides health care, more and more products prolongingyouth and acting against aging come to market each year. Formost of them, clear scientific demonstration of effectivenessis insufficient or even absent.

In vivo tests with cell cultures are expensive, as well thosewith animals that, in addition, are rejected by an increasingpart of the society for ethical aspects.

For many years yeast has been used as a simple modelfor the study of basic mechanism underlying the complexphenomena occurring in higher eukaryotes.

Among many other advantages, the knowledge of theentire genome and the availability of mutants in each of thenear six thousand genes render this organism a powerful toolfor basal as well as for applicative studies.

In this respect, we successfully used yeast to evaluatethe toxicity of new arylthioindoles (ATIs), which are potenttubulin assembly inhibitors [22, 23].


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Figure 2: Effect of apple components on the production of intracellular ROS during aging. MCY4/Kllsm4::KanMX4 yeast cells weregrown in the presence of extracts from the peel, flesh, or entire apple and the polyphenolic fraction. Acetone was used as a control. Cellsfrom exponential (EXP; (a)) and stationary (STAT; (b)) cultures were incubated with DHR123 and after 3 h observed at the fluorescencemicroscope. In (c) is reported the percentage of ROS positive cells. Average and standard deviations, obtained from three independentexperiments, are also indicated.


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Figure 3: Effect of apple components on the oxidative stressresistance. Viability of the MCY4/Kllsm4Δ1 strain was measuredafter exposure of cells to H2O2 at the indicated concentrationfor 4 h in the presence of acetone (control) or extracts fromwhole apple, skin, flesh, and polyphenolic fractions. Average andstandard deviations, obtained from three independent experiments,are indicated.

In another work, by the use of a particular strain of S.cerevisiae, mutated in the LSM4 gene and showing prematureaging and loss of viability, we demonstrated that carnitines,in particular acetyl-L-carnitine (ALC), are able to prolonglifespan, to protect cells from oxidative stress and preventmitochondrial fragmentation [13].

LSM4 is an essential gene encoding a subunit of theLsm complex involved in mRNA decapping and splicing[17]. In previous works we demonstrated that S. cerevisiaestrains not expressing the endogenous Lsm4p recoveredviability when transformed with Kllsm4, the hortolog genefrom Kluyveromyces lactis, as well with a truncated form ofthis gene (Kllsm4Δ1) still containing the Sm-like domainsThe Kllsm4Δ1 mutants showed accumulation of mRNAdegradation intermediates, the increase of intracellular ROS,undergo cell death prematurely during stationary phase andshow the typical markers of apoptosis. A quite normalcell viability observed in one of these mutants (lsm4) wasrecovered following the deletion of the yeastmeta-caspaseYCA1 gene, suggesting that caspase activity is required forapoptosis induced by increased mRNA stability [16, 24, 25].

The short lifespan and the high sensitivity to oxidativestress of Kllsm4Δ1 mutants, compared to wild-type yeaststrains, render them good candidates to test the ability ofsubstances to prevent aging and cell death.

In this paper, we used this particular mutant to study thecapability of apple’s solution and derived fractions to increaseviability and oxidative stress resistance in yeast cells.

Our result show that solutions of entire apples canprolong cellular life span by 100%, more than any singleapple fractions. In fact, while flesh showed a modest effect,

skin increased life span by 40%, probably due to its contentof antioxidant polyphenols, as also suggested by the 60%increase of viability observed with cultures treated withpurified polyphenol fractions. The fact that the purifiedpolyphenols had a quite high activity, speaks in favour ofan important role of apple polyphenols. However, sincethe whole apple extract had superior activity in spite of amuch lower content of polyphenols in respect of the skinextract suggested that compounds other than polyphenolsare contributing to the observed extension of the cellular lifespan.

Similarly, more than skin and polyphenols, entire applesstrongly reduced ROS production in both exponential andstationary aging cells. In contrast, apple flesh stronglyincreased ROS production, especially in stationary phasecells.

Since flesh shows a very low positive effect on cellviability, one can conclude that the potential toxicity of theobserved increase of ROS is in some way neutralized by othercomponents present in the entire apple.

Whole apple extracts, closely followed by the polyphenolfraction, can also protect cells efficiently from hydrogenperoxide treatments while skin and pulp have a minor effectslightly varying with H2O2 concentration.

These results, in their substance, were confirmed whenthe same fractions were included into a creamy matrix, withthe entire apple homogenate showing the best protection.

These findings are in agreement with Vanzani et al.[7] who reported that the antioxidant efficiency of thepure apple compounds was lower than that of the appleextracts, and that the higher efficiency of apples appears to bestrictly related to the overwhelming presence of oligomericproanthocyanidins.

In conclusion, the wide knowledge of yeast and theavailability of genetic, biochemical and molecular biologytools make this organism a simple system useful for assayingin vivo the effects, among others, of anti-aging molecules.The promising results of this preliminary study provide thebasis to plan further researches aimed to precisely identifyingthe apple compounds capable to modulate the life-extendingproperties of apple on yeasts.

4. Materials and Methods

4.1. Yeast Strains and Growth Conditions. We used the S.cerevisiae strains MCY4/KlLSM4::kanMX4 (MAT α, ade1-101, his3-Δ1, trp1-289, ura3, LEU-GAL1-SDB23, pRS416/Kllsm4Δ1) [14] and MCY4/313Kllsm4Δ1 (Mat α, ade1-101, his3-Δ1, trp1-289, ura3, LEU2-GAL1-SDB23, pRS313/Kllsm4Δ1) [16].

Cells were grown in YP (1% yeast extract, 2% peptone)supplemented with 2% glucose (YPD) at 28◦C with theaddition of or in SD (yeast nitrogen base without amino acidsand auxotrophic requirement as needed). Solid media weresupplemented with 2% Bactoagar (Difco, Detroit, MI, USA).

4.2. Cell Viability. The determination of chronological lifes-pan was done as described in Palermo et al., 2007 [26].Briefly, cell suspensions (5 μL) containing approximately


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Figure 4: Effect of apple solutions included in ointment. (a) Cells of strain MCY4/Kllsm4::KanMX4 were grown in YPD+G418 antibiotic toprevent contaminations. Ointment base Essex was used as a control. Viability was monitored as percentage of microcolony forming units.Standard deviation was obtained from three independent experiments. (b) 10-fold serial dilutions of each cell culture described above werespotted onto YPD plates (1% bactopeptone, 1% yeast extract, 2% glucose, and 2% bacto agar) and incubated 2 days before recording. (c)Cells of strain MCY4/Kllsm4::KanMX4 were grown in YPD + G418 antibiotic in the presence of apple solutions in ointment and exposed toH2O2 at the indicated concentration for 4 h. Average and standard deviations, obtained from three independent experiments are indicated.

6 · 106 cells mL−1 were poured on a thin layer of YPD agar ona microscope slide. A cover slip was placed over the samplesand, after 24 h, viable and unviable cells were scored on thebasis of their ability to form microcolonies.

Cells were grown at 28◦C in minimal medium (0,67%yeast nitrogen base without amino acids) containing 2%glucose (SD) supplemented with 20 microg/mL of the appro-priate nutritional requirements according to the genotype ofthe strains, or in YPD + G418.

For the viability spot assay, cell suspensions at theconcentration of 107 cells/mL were transferred in microtiterplates, serially ten-fold diluted and spotted onto YP plates(1% yeast extract, 2% peptone) supplemented with 2%

glucose (YPD). Plates were incubated at 28◦C for five daysbefore recording.

4.3. H2O2 Sensitivity. To determine the sensitivity to oxygenperoxide, cells growing exponentially were exposed to 0.8,1.2, and 3 mM H2O2 at 28◦C for 4 h. Cell viability wasdetermined by counting the formation of microcolonies.

4.4. ROS Detection. Dihydrorhodamine 123 (Sigma) wasadded at a concentration of 5 μg/mL of cell culture from a2.5 mg/mL stock solution in ethanol and cells were viewedwithout further processing through a rhodamine opticalfilter after 3 h incubation [27].


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Figure 5: Comparison of the average concentrations (expressed aschromatographic area) of some characteristic apple polyphenols inthe extracts used for the viability tests. The vertical bars representthe standard deviation of the measure.

4.5. Sample Preparation

Extracts. Apples, from the cv. Golden Delicious, produced inTrentino, Italy, were purchased directly from the producersin June 2009, and extracted as described in Vrhovsek [6]. Tolimit the enzymatic oxidation and chemical reactions, boththe apples and the extraction solution were cooled to 4◦C.Each sample was prepared out of three fruits. The core wasremoved with a corer, in order to study only the edible part.

Whole Apple Extract. Each apple was cut into equal slices.Three slices (cortex + skin) from the opposite side of eachfruit (total weight 400 g) were used for the preparation ofaqueous acetone extracts. The sample was homogenized ina blender Osterizer mod. 847–86 at speed one in 1500 mL ofmixture acetone/water (70/30 w/w), to produce a raw extractcorresponding to 26.7 g of fresh apple in 100 mL.

Whole Apple Polyphenols. An aliquot of 750 mL of theabove extract, corresponding to 200 g of apple, was used toprepare a partially purified extract, containing most applepolyphenols. Acetone was removed by evaporation underreduced pressure at 35◦C, from a volume of 200 mL ofapple polyphenols extract, which was brought to 100 mLwith water. This extract was loaded on a preparative columncontaining 20 g of Isolute ENV + resin (Biotage, Uppsala,Sweden), previously activated with 300 mL MeOH and400 mL water. All the hydrophilic compounds were washedwith 300 mL of water, then the polyphenols were eluted with400 mL of methanol. After the whole extract was processed,the methanol was removed by evaporation under reducedpressure, and the purified fraction was redissolved into750 mL of acetone/water (70/30 w/w).

Skin Extract. Each apple was manually peeled. An aliquotof 40 g of apple skins was extracted with 166 mL of themixture acetone/water (70/30 w/w), to produce a raw extractcorresponding to 24.0 g of apple skins in 100 mL.

Flesh Extract. Each apple was manually peeled. An aliquot of200 g of apple flesh (cortex) was extracted with 750 mL of themixture acetone/water (70/30 w/w), to produce a raw extractcorresponding to 26.7 g of apple cortex in 100 mL.

The centrifuged extracts were stored at−20◦C for the cellviability trials and analysis of total polyphenols.

Ointments. Fresh apples were crushed and filtered. The clearliquid (20 mL) was added to ointment base Essex (80 g) whilestirring.

4.6. Chemical Analyses

Spectrophotometric Analysis of Total Polyphenols. The totalamount of polyphenols was quantified with an optimizedFolin-Ciocalteu method [28] according to which interferingcompounds such as sugars, amino acids, and ascorbic acidwere removed by cleanup on a C-18 cartridge (0.5 g, Seppak, Waters) from the sample reconstituted in water asdescribed above. The results are expressed as equivalent of(+)-catechin, mg/kg of FW.

Fingerprinting of the Extracts by LCMS. Analysis was carriedout using a Waters Acquity UPLC, coupled to a SynaptHDMS QTOF-MS (Waters, Manchester, UK) via an electro-spray interface (ESI), operating in W-mode. The softwareused was Masslynx 4.1. The reverse phase method wasperformed on a ACQUITY UPLC 1.8 μm 2.1 × 150 mm HSST3 (Waters) column, maintained at 30◦C for 30 min using0.1% formic acid in water as solvent A and 0.1% formic acidin methanol as solvent B with the following gradient: until6 min isocratic at 100% A, then increasing linearly to 100% Bat 26 min and held isocratic at 100% B till 30 min. After eachrun, the column was brought the initial conditions (30% ofsolvent A) in 3 min.

Spectra were collected in negative ESI mode over a massrange 50–3000 amu with a scan duration of 0.3 s in centroidmode. The transfer collision energy and trap collision energywere set at 4 and 6 V, respectively. The source parameterswere: capillary 2.5 kV, sampling cone 25 V, extraction cone3 V, source temperature 150◦C, desolvation temperature500◦C, desolvation gas flow 1000 l/h, and nebulizer gas flow50 l/h. External calibration of the instrument was performedat the beginning of each batch of analysis by direct infusionof a sodium formate solution (10% formic acid/0.1 MNaOH/Acetonitrile at a ratio of 1/1/8) by controlling themass accuracy (less than 5 ppm) and mass resolution (over14000 FWHM). LockMass calibration was applied using asolution of leucine enkephaline (0.5 mg/L, m/z 556.2771 forpositive and 554.2620 for negative ion mode) at 0.1 mL/min.

Each extract was analysed 10 times. The relative concen-tration of major apple polyphenols in the extracts are givenin Figure 5.

Acknowledgments

Domenico Masuero is acknowledged for excellent technical


support in the spectrophotometric and LCMS analysis. Thiswork was supported by PRIN 2009.

References

[1] R. Velasco, A. Zharkikh, J. Affourtit et al., “The genome ofthe domesticated apple (Malus × domestica Borkh.),” NatureGenetics, vol. 42, no. 10, pp. 833–839, 2010.

[2] S. Reagan-Shaw, D. Eggert, H. Mukhtar, and N. Ahmad,“Antiproliferative effects of apple peel extract against cancercells,” Nutrition and Cancer, vol. 62, no. 4, pp. 517–524, 2010.

[3] C. Gerhauser, “Cancer chemopreventive potential of apples,apple juice, and apple components,” Planta Medica, vol. 74,no. 13, pp. 1608–1624, 2008.

[4] J. Boyer and R. H. Liu, “Apple phytochemicals and their healthbenefits,” Nutrition Journal, vol. 3, article no. 1, pp. 1–45, 2004.

[5] T. K. McGhie, S. Hudault, R. C. M. Lunken, and J. T.Christeller, “Apple peels, from seven cultivars, have lipase-inhibitory activity and contain numerous ursenoic acids asidentified by LC-ESI-QTOF-HRMS,” Journal of Agriculturaland Food Chemistry, vol. 60, no. 1, pp. 482–491, 2012.

[6] U. Vrhovsek, A. Rigo, D. Tonon, and F. Mattivi, “Quantitationof polyphenols in different apple varieties,” Journal of Agricul-tural and Food Chemistry, vol. 52, no. 21, pp. 6532–6538, 2004.

[7] P. Vanzani, M. Rossetto, A. Rigo et al., “Major phytochemicalsin apple cultivars: contribution to peroxyl radical trappingefficiency,” Journal of Agricultural and Food Chemistry, vol. 53,no. 9, pp. 3377–3382, 2005.

[8] R. H. Liu and J. Sun, “Antiproliferative activity of apples isnot due to phenolic-induced hydrogen peroxide formation,”Journal of Agricultural and Food Chemistry, vol. 51, no. 6, pp.1718–1723, 2003.

[9] M. Waldecker, T. Kautenburger, H. Daumann et al., “Histone-deacetylase inhibition and butyrate formation: fecal slurryincubations with apple pectin and apple juice extracts,”Nutrition, vol. 24, no. 4, pp. 366–374, 2008.

[10] M. G. Mirisola and V. D. Longo, “Conserved role of Ras-GEFsin promoting aging: from yeast to mice,” Aging, vol. 3, pp. 340–343, 2011.

[11] D. Carmona-Gutierrez, T. Eisenberg, S. Buttner, C. Meisinger,G. Kroemer, and F. Madeo, “Apoptosis in yeast: triggers,pathways, subroutines,” Cell Death and Differentiation, vol. 17,no. 5, pp. 763–773, 2010.

[12] A. Cassidy-Stone, J. E. Chipuk, E. Ingerman et al., “Chemicalinhibition of the mitochondrial division dynamin reveals itsrole in bax/bak-dependent mitochondrial outer membranepermeabilization,” Developmental Cell, vol. 14, no. 2, pp. 193–204, 2008.

[13] V. Palermo, C. Falcone, M. Calvani, and C. Mazzoni, “Acetyl-L-carnitine protects yeast cells from apoptosis and aging andinhibits mitochondrial fission.,” Aging cell, vol. 9, no. 4, pp.570–579, 2010.

[14] C. Mazzoni and C. Falcone, “Isolation and study of KILSM4,a Kluyveromyces lactis gene homologous to the essential geneLSM4 of Saccharomyces cerevisiae,” Yeast, vol. 18, no. 13, pp.1249–1256, 2001.

[15] C. Mazzoni, P. Mancini, F. Madeo, V. Palermo, and C. Falcone,“A Kluyveromyces lactis mutant in the essential gene KlLSM4shows phenotypic markers of apoptosis,” FEMS Yeast Research,vol. 4, no. 1, pp. 29–35, 2003.

[16] C. Mazzoni, E. Herker, V. Palermo et al., “Yeast caspase 1 linksmessenger RNA stability to apoptosis in yeast,” EMBO Reports,vol. 6, no. 11, pp. 1076–1081, 2005.

[17] M. Cooper, L. H. Johnston, and J. D. Beggs, “Identificationand characterization of Uss1p (Sdb23p): a novel U6 snRNA-associated protein with significant similarity to core proteinsof small nuclear ribonucleoproteins,” EMBO Journal, vol. 14,no. 9, pp. 2066–2075, 1995.

[18] A. E. Mayes, L. Verdone, P. Legrain, and J. D. Beggs, “Char-acterization of Sm-like proteins in yeast and their associationwith U6 snRNA,” EMBO Journal, vol. 18, no. 15, pp. 4321–4331, 1999.

[19] C. Mazzoni, P. Mancini, L. Verdone et al., “A truncated formof K1Lsm4p and the absence of factors involved in mRNAdecapping trigger apoptosis in yeast,” Molecular Biology of theCell, vol. 14, no. 2, pp. 721–729, 2003.

[20] F. Chinnici, A. Bendini, A. Gaiani, and C. Riponi, “Radicalscavenging activities of peels and pulps from cv. goldendelicious apples as related to their phenolic composition,”Journal of Agricultural and Food Chemistry, vol. 52, no. 15, pp.4684–4689, 2004.

[21] P. Franceschi, Y. Dong, K. Strupat, U. Vrhovsek, and F. Mattivi,“Combining intensity correlation analysis and MALDI imag-ing to study the distribution of flavonols and dihydrochalconesin Golden Delicious apples,” Journal of Experimental Botany,vol. 63, no. 3, pp. 1123–1133, 2012.

[22] G. La Regina, T. Sarkar, R. Bai et al., “New arylthioindoles andrelated bioisosteres at the sulfur bridging group. 4. Synthesis,tubulin polymerization, cell growth inhibition, and molecularmodeling studies,” Journal of Medicinal Chemistry, vol. 52, no.23, pp. 7512–7527, 2009.

[23] V. Palermo, L. Pieri, R. Silvestri, G. La Regina, C. Falcone, andC. Mazzoni, “Drug-induced inhibition of tubulin polymeriza-tion induces mitochondrion-mediated apoptosis in yeast,” CellCycle, vol. 10, no. 18, pp. 3208–3209, 2011.

[24] C. Mazzoni and C. Falcone, “mRNA stability and control ofcell proliferation,” Biochemical Society Transactions, vol. 39, no.5, pp. 1461–1465, 2011.

[25] C. Mazzoni and C. Falcone, “Caspase-dependent apoptosisin yeast,” Biochimica et Biophysica Acta, vol. 1783, no. 7, pp.1320–1327, 2008.

[26] V. Palermo, C. Falcone, and C. Mazzoni, “Apoptosis and agingin mitochondrial morphology mutants of S. cerevisiae,” FoliaMicrobiologica, vol. 52, no. 5, pp. 479–483, 2007.

[27] F. Madeo, E. Frohlich, M. Ligr et al., “Oxygen stress: a regulatorof apoptosis in yeast,” Journal of Cell Biology, vol. 145, no. 4,pp. 757–767, 1999.

[28] A. Rigo, F. Vianello, G. Clementi et al., “Contribution ofproanthocyanidins to the peroxy radical scavenging capacityof some Italian red wines,” Journal of Agricultural and FoodChemistry, vol. 48, no. 6, pp. 1996–2002, 2000.

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